T-CELL ACTIVATION-DEPENDENT ASSOCIATION BETWEEN THE P85 SUBUNIT OF THE PHOSPHATIDYLINOSITOL 3-KINASE AND GRB2/PHOSPHOLIPASE C-GAMMA-1-BINDING PHOSPHOTYROSYL PROTEIN PP36/38

Tyrosine phosphorylation of cellular proteins is an early and an essential step in T cell receptor-mediated lymphocyte activation, Tyrosine phosphorylation of transmembrane receptor chains (such as zeta and CD3 chains) and membrane-associated proteins provides docking sites for SH2 domains of adaptor proteins and signaling enzymes, resulting in their recruitment in the vicinity of activated receptors, pp36/38 is a prominent substrate of early tyrosine phosphorylation upon stimulation through the T cell receptor, The tyrosine-phosphorylated form of pp36/38 is membrane-associated and directly interacts with phospholipase C-gamma l and Grb2, providing one mechanism to recruit downstream effecters to the cell membrane, Here, we demonstrate that in Jurkat T cells, pp36/38 associates with the p85 subunit of phosphatidylinositol 3-kinase (PI-S-K p85) in an activation-dependent manner, Association of pp36/38 with PI-3-K p85 was confirmed by transfection of a hemagglutinin-tagged p85 alpha cDNA into Jurkat cells followed by anti-hemagglutinin immunoprecipitation, In vitro binding experiments with glutathione S-transferase fusion proteins of PI-S-K p85 demonstrated that the SH2 domains, but not the SH3 domain, mediated binding to pp36/38, This binding was selectively abrogated by phosphopeptides that bind to p85 SH2 domains with high affinity, Filter binding assays demonstrated that association between pp36/38 and PI-3-K p85 SH2 domains was due to direct binding. These results strongly suggest the role of pp36/38 in recruiting PI-3-K to the cell membrane and further support the idea that pp36/38 is a multifunctional docking protein for SH2 domain-containing signaling proteins in T cells.